Methods of using sustained release aminopyridine compositions

ABSTRACT

A pharmaceutical composition which comprises a therapeutically effective amount of a aminopyridine dispersed in a release matrix, including, for example, a composition that can be formulated into a stable, sustained-release oral dosage formulation, such as a tablet which provides, upon administration to a patient, a therapeutically effective plasma level of the aminopyridine for a period of at about 12 hours and the use of the composition to treat various neurological diseases, including multiple sclerosis. A method of selecting individuals based on responsiveness to a treatment, including, for example, identifying individuals who responded to treatment with a sustained release fampridine composition.

CROSS REFERENCES

This application claims priority to U.S. Provisional Application No.60/560,894 filed Apr. 9, 2004.

BACKGROUND

This invention relates a sustained release oral dosage form of anaminopyridine pharmaceutical composition that can be used to treatindividuals affected with neurological disorders wherein saidpharmaceutical composition maximizes the therapeutic effect, whileminimizing adverse side effects.

The sustained release oral dosage form of the present invention may beutilized to treat neurological disorders such as multiple sclerosis,spinal cord injuries, Alzheimer's disease and ALS.

Multiple sclerosis (MS) is a degenerative and inflammatory neurologicaldisease that affects the central nervous system, more specifically themyelin sheath. The condition of MS involves demyelination of nervefibers resulting in “short-circuiting” of nerve impulses and thus aslowing or blocking of transmission along the nerve fibers, withassociated disabling symptoms. Treatment alternatives for promotingtransmission along affected nerves have thus far been limited.

Potassium channel blockers are a class of compounds that has been foundto improve the conduction of nerve impulses. As a result, they havebecome the focus of attention in the symptomatic treatment of spinalcord injury, MS and Alzheimer's disease. One sub-class of potassiumchannel blockers, aminopyridines have shown promise in the treatment ofneurological diseases. 4-aminopyridine (4-AP), a mono-aminopyridineknown as fampridine, has been found to reduce the potassium flow innerve impulse transmission and, thereby, shows effectiveness inrestoring conduction in blocked and demyelinated nerves.

Early studies of monoaminopyridines were conducted using an intravenouscomposition, comprising 4-AP. This was followed by the development of animmediate-release (IR) composition for oral administration of 4-AP,commonly known as fampridine. The IR composition consisted of 4-APpowder in a gelatin-based capsule and produced rapid peak plasmaconcentrations shortly after dosing with a time to maximum concentrationof about 1 hour and a plasma half life of about 3.5 hours. The rapidrelease and short half life of fampridine makes it difficult to maintaineffective plasma levels without producing high peaks following each dosethat may cause undesirable side effects such as seizures and trembling.

Electrophysiological recordings from isolated spinal cord have shownchronic failure of action potential conduction in surviving myelinatedaxons, following a blunt contusion injury (Blight, A. R., “Axonalphysiology of chronic spinal cord injury in the cat: intracellularrecording in vitro”, Neuroscience. 10:1471-1486 (1983b)). Some of thisconduction block can be overcome, at the level of single nerve fibers,using the drug 4-aminopyridine (4-AP) (Blight, A. R., “Effect of4-aminopyridine on axonal conduction-block in chronic spinal cordinjury”, Brain Res. Bull. 22:47-52 (1989)). Intravenous injection ofthis compound in animals with experimental or naturally occurring spinalcord injuries produces significant improvements in electrophysiological(Blight, A. R. and Gruner, J. A., “Augmentation by 4-aminopyridine ofvestibulospinal free fall responses in chronic spinal-injured cats,” J.Neurol. Sci. 82:145-159, (1987)) and behavior function (Blight, A. R.,“The effects of 4-aminopyridine on neurological deficits in chroniccases of traumatic spinal cord injury in dogs: a phase I clinicaltrial,” J. Neurotrauma, 8:103-119 (1991)).

An initial study in spinal cord injury patients was organized by Dr.Keith Hayes and indicated a potential for a therapeutic benefit, mostlyat the electrophysiological level, combined with a lack of serious sideeffects (Hayes et al, “Effects of intravenous 4-aminopyridine onneurological function in chronic spinal cord injured patients:preliminary observations,” Proc. IBRO World Conf. Neurosci., p. 3451991).

A recent study of fampridine in patients with chronic incomplete SCI wasreported in Clinical Neuropharmacology 2003: Keith C. Hayes; Patrick J.Potter; Robert R. Hansebout; Joanne M. Bugaresti; Jane T. C. Hsieh; SeraNicosia; Mitchell A. Katz; Andrew R. Blight; Ron Cohen 26(4):185-192.

SUMMARY OF THE INVENTION

One embodiment of the present invention relates to a pharmaceuticalcomposition which contains one or more potassium channel blockers andwhich can be used in the effective treatment of various diseases, forexample, spinal cord injury, multiple sclerosis, Alzheimer's disease,and ALS. Embodiments of the present invention are directed tocompositions that include a matrix and a potassium channel blocker. Thepotassium channel blockers may include aminopyridines, for example,4-aminopyridine, 3,4-diaminopyridine and the like, most preferably4-aminopyridine. The composition provides for sustained-release of theaminopyridine from the matrix to maintain the efficacious and safeplasma level of an aminopyridine. The aminopyridine dispersed in thematrix is capable of providing, upon administration to a patient, adesired release profile. The composition may be used to establish inpatients in need of such treatment, a therapeutically effective bloodplasma level of the aminopyridine for a period of at least about 6 hoursand preferably up to at least about 12 hours in the patient in atwice-daily administration while avoiding excessive peaks and troughs inthe level of the aminopyridine. The composition may include a mono- ordi-aminopyridine, preferably 4-AP or 3,4-DAP or a combination thereof,homogeneously dispersed in a rate-controlling polymer matrix, preferablyincluding a hydrophilic polymer like hydroxypropylmethylcellulose(HPMC). The composition of the present invention may also include one ormore additional active ingredients and/or one or more pharmaceuticallyacceptable excipients. These compositions can be used to treat variousneurological diseases, for example, spinal cord injury, multiplesclerosis, Alzheimer's disease, and ALS.

Another embodiment of the present invention is a stable pharmaceuticalcomposition that comprises a therapeutically effective amount of anaminopyridine dispersed in a matrix that provides a release profile ofthe aminopyridine to a patient that has a desired C_(max) to C_(τ)ratio. The composition may be used to establish and/or maintain in apatient, a therapeutically effective level of the aminopyridine.Preferably the aminopyridine in the composition is released over time sothat a therapeutically effective level of the aminopyridine in thepatient can be achieved with twice daily dosing of the composition. In amore preferred embodiment, undesirable spikes or peaks in the release ofthe aminopyridine are avoided.

Another embodiment of the present invention is a stable,sustained-release oral dosage formulation of a composition whichincludes a therapeutically effective amount of a 4-aminopyridinedispersed in a matrix that provides a release profile of 4-aminopyridinein the blood plasma of the patient extending over a period of at least 6hours, preferably at least 8 hours, and more preferably, at least about12 hours. In another embodiment, a stable, sustained-release oral dosageformulation of a composition includes a therapeutically effective amountof a 4-aminopyridine dispersed in a matrix that provides atherapeutically effective blood plasma level of 4-aminopyridine in thepatient extending over about 24 hours.

Preferably, the oral dosage formulation of the composition is amonolithic tablet formed by compression of the pharmaceuticalcomposition of the present invention. In preferred embodiments, the oraldosage formulation includes a compressed tablet of a therapeuticallyeffective amount of 4-aminopyridine dispersed in matrix that includes ahydrophilic polymer such as HPMC. The oral dosage form of the presentinvention may also include one or more pharmaceutically acceptableexcipients.

The dispersion of 4-aminopyridine throughout the matrix imparts chemicaland physical stability to the composition while providing asustained-release profile. This enhanced dosage stability is mostnotably observed in compositions and dosage forms of the presentinvention having low concentrations of 4-aminopyridine, and stability isachieved while maintaining the desired controlled-release profile.Specifically, the compressed tablet formulation of the present inventionexhibits superior resistance to moisture absorption by ambient humidityand maintains a uniform distribution of the 4-aminopyridine throughoutthe tablet while providing a release profile of 4-aminopyridine thatpermits establishment of a therapeutically effective concentration ofthe potassium channel blocker with once daily or twice daily dosing ofthe formulation. Preferably the therapeutically effective concentrationreleased by the formulation extends over at least about 6 hours,preferably at least about 8 hours, and more preferably at least about 12hours. In addition, the homogeneity of the dosage form renders itamenable to formation by simple and inexpensive manufacturing processesas compared with the multi-layered structure of prior sustained-releasedosage formulations.

The compositions of the present invention may be used in the treatmentof a condition in a patient that includes establishing a therapeuticallyeffective concentration of a potassium channel blocker in the patient inneed thereof. The compositions may be used for building up a level andor maintaining a therapeutically effective concentration of anaminopyridine in the patient by twice daily dosing. The dosages of thepresent compositions can be made with a lower concentration of theaminopyridine to facilitate restful periods for the patient during theday or night, depending on desired results or dosage schedule. Wheredesirable, the compositions of the present invention may be formulatedto avoid large peaks in initial release of the aminopyridine. Thecompositions of the present invention when administered to a patient inneed thereof provide for the treatment of neurological diseases that arecharacterized by a degradation of nerve impulse transmission.Preferably, the compositions are a stable, sustained-release tablet of atherapeutically effective amount of a mono- or di-aminopyridine,dispersed in HPMC such that therapeutically effective blood plasma levelof the mono- or di-aminopyridine is maintained in the patient for aperiod of at least 6 hours, preferably at least 8 hours, and morepreferably at least about 10-12 hours in a once or twice dailyadministration.

One embodiment of the present invention relates to a method ofincreasing walking speed comprising administering to a patient withmultiple sclerosis an effective amount of a sustained releaseaminopyridine composition twice daily, wherein said effective amount isless than about 15 milligrams of aminopyridine. In a preferredembodiment, the effective amount is about 10 to about 15 milligrams ofaminopyridine.

In a further embodiment of the present invention a method of improvinglower extremity muscle tone comprising administering to a patient withmultiple sclerosis an effective amount of a sustained-releaseaminopyridine composition twice daily is provided. In a preferredembodiment, said effective amount is less than about 15 milligrams ofaminopyridine.

Another embodiment of the present invention relates to a method ofimproving lower extremity muscle strength comprising administering to apatient with multiple sclerosis an effective amount of asustained-release aminopyridine composition twice daily, wherein saideffective amount is less than about 15 milligrams of aminopyridine.

One embodiment of the present invention relates to a method of selectingindividuals based on responsiveness to a treatment. The method comprisesidentifying a plurality of individuals; administering a test to eachindividual prior to a treatment period; administering a treatment to oneor more of the individuals during the treatment period; administeringthe test a plurality of times to each individual during the treatmentperiod; and selecting one or more individuals, wherein the selectedindividuals exhibit an improved performance during a majority of thetests administered during the treatment period as compared to the testadministered prior to the treatment period. In certain embodiments, themethod may further comprise administering the test to each individualafter the treatment period, wherein the selected individuals furtherexhibit an improved performance during a majority of the testsadministered during the treatment period as compared to the testadministered after the treatment period.

A further embodiment relates to a method of selecting individuals basedon responsiveness to a treatment, the method comprising identifying aplurality of individuals; administering a test to each individual priorto a treatment period; administering a treatment to one or more of theindividuals during the treatment period; administering the test aplurality of times to each individual during the treatment period;administering the test to each individual after the treatment period;and selecting one or more individuals, wherein the selected individualsexhibit an improved performance during a majority of the testsadministered during the treatment period as compared to the betterperformance of the test administered prior to the treatment period andthe test administered after the treatment period.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a histogram to show the number of treatment visits at whichsubjects showed faster walking speed on the timed 25 foot walk than atall of the five non-treatment visits.

FIG. 2 is a graph of the average walking speeds (ft/sec) by study day(observed cases, ITT population).

FIG. 3 is a histogram of the percent change in average walking speedduring the 12-week stable dose period (observed cases, ITT population).

FIG. 4 is a histogram of the percentage of protocol specified responders(subjects with average changes in walking speed during the 12-weekstable dose period of at least 20%) by treatment group [(observed cases,ITT population]).

FIG. 5 is a graph of LEMMT by study day (observed cases, ITTpopulation).

FIG. 6 is a histogram of change in LEMMT during the 12-week stable doseperiod (observed cases, ITT population).

FIG. 7 is a histogram of the percentage of post hoc responders bytreatment group TIT population) according to a responder analysis of thepresent invention.

FIG. 8 is a histogram of the percentage of responders for placebosubjects vs. fampridine subjects pooled (ITT population) according to aresponder analysis of the present invention.

FIG. 9 are histograms of the validation of the post hoc respondervariable using subjective scales (observed cases, ITT population).

FIG. 10 is a graph of percent change in walking speed at eachdouble-blind visit by responder analysis grouping (observed cases, ITTpopulation).

FIG. 11 is a graph of the change in LEMMT at each double-blind visit byresponder analysis grouping (observed cases, ITT population).

FIG. 12 is a graph of change in overall Ashworth Score at eachdouble-blind visit by responder analysis grouping (observed cases, ITTpopulation).

DETAILED DESCRIPTION OF THE INVENTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularmolecules, compositions, methodologies or protocols described, as thesemay vary. It is also to be understood that the terminology used in thedescription is for the purpose of describing the particular versions orembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

The terms used herein have meanings recognized and known to those ofskill in the art, however, for convenience and completeness, particularterms and their meanings are set forth below.

It must also be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference toa “spheroid” is a reference to one or more spheroid and equivalentsthereof known to those skilled in the art, and so forth. Unless definedotherwise, all technical and scientific terms used herein have the samemeanings as commonly understood by one of ordinary skill in the art.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare now described. All publications mentioned herein are incorporated byreference. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

“Local administration” means direct administration by a non-systemicroute at or in the vicinity of the site of affliction, disorder, orperceived pain.

The terms “patient” and “subject” mean all animals including humans.Examples of patients or subjects include humans, cows, dogs, cats,goats, sheep, and pigs.

The term “pharmaceutically acceptable salts, esters, amides, andprodrugs” as used herein refers to those carboxylate salts, amino acidaddition salts, esters, amides, and prodrugs of the compounds of thepresent invention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of patients without unduetoxicity, irritation, allergic response, and the like, commensurate witha reasonable benefit/risk ratio, and effective for their intended use,as well as the zwitterionic forms, where possible, of the compounds ofthe invention.

The term “prodrug” refers to compounds that are rapidly transformed invivo to yield the parent compounds of the above formula, for example, byhydrolysis in blood. A thorough discussion is provided in T. Higuchi andV. Stella, “Pro-drugs as Novel Delivery Systems,” Vol. 14 of the A.C.S.Symposium Series, and in Bioreversible Carriers in Drug Design, ed.Edward B. Roche, American Pharmaceutical Association and Pergamon Press,1987, both of which are incorporated herein by reference.

The term “salts” refers to the relatively non-toxic, inorganic andorganic acid addition salts of compounds of the present invention. Thesesalts can be prepared in situ during the final isolation andpurification of the compounds or by separately reacting the purifiedcompound in its free base form with a suitable organic or inorganic acidand isolating the salt thus formed. Representative salts include thehydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate,oxalate, valerate, oleate, palmitate, stearate, laurate, borate,benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionateand laurylsulphonate salts, and the like. These may include cationsbased on the alkali and alkaline earth metals, such as sodium, lithium,potassium, calcium, magnesium, and the like, as well as non-toxicammonium, tetramethylammonium, tetramethylammonium, methlyamine,dimethlyamine, trimethlyamine, triethlyamine, ethylamine, and the like.(See, for example, S. M. Barge et al., “Pharmaceutical Salts,” J. Pharm.Sci., 1977, 66:1-19 which is incorporated herein by reference.).

A “therapeutically effective amount” is an amount sufficient to decreaseor prevent the symptoms associated with a medical condition orinfirmity, to normalize body functions in disease or disorders thatresult in impairment of specific bodily functions, or to provideimprovement in one or more of the clinically measured parameters of thedisease. Preferably, improvement in symptoms associated with the diseaseincluding walking speed, lower extremity muscle tone, lower extremitymuscle strength, or spasticity. As related to the present application, atherapeutically effective amount is an amount sufficient to reduce thepain or spasticity associated with the neurological disorder beingtreated, or an amount sufficient to result in improvement of sexual,bladder or bowel function in subjects having a neurological disorderwhich impairs nerve conduction, which hinders normal sexual, bladder orbowel functions.

“Treatment” refers to the administration of medicine or the performanceof medical procedures with respect to a patient, for either prophylaxis(prevention), to cure the infirmity or malady in the instance where thepatient is afflicted refers, or amelioration the clinical condition ofthe patient, including a decreased duration of illness or severity ofillness, or subjective improvement in the quality of life of the patientor a prolonged survival of the patient.

In addition, the compounds of the present invention can exist inunsolvated as well as solvated forms with pharmaceutically acceptablesolvents such as water, ethanol, and the like. In general, the solvatedforms are considered equivalent to the unsolvated forms for the purposesof the present invention.

One aspect of the invention is a sustained-release pharmaceuticalcomposition comprising an aminopyridine dispersed in a sustained releasematrix such as a rate-controlling polymer. The composition of thepresent invention is capable of providing, upon administration to apatient, a release profile of the aminopyridine extending over at least6 hours, preferably least about 12 hours, and more preferably at least24 hours or more. Preferably the aminopyridine concentration in thecomposition is a therapeutically effective amount, and preferably theaminopyridine is dispersed uniformly throughout the release matrix. Atherapeutically effective amount is an amount of a potassium channelblocker, preferably an aminopyridine compound, that when administered toa patient or subject, ameliorates a symptom of a neurological disease.

When the compositions of the present invention are administered to apatient, the concentration of the aminopyridine in the patient's plasmaover time (release profile) may extend over a period of at least 6hours, preferably over at least 8 hours, and more preferably over atabout 12 hours. The compositions may provide in single dose a meanmaximum plasma concentration of aminopyridine in the patient of fromabout 15 to about 180 ng/ml; a mean T_(max) from about 1 to about 6hours, more preferably about 2 to about 5.2 hours after administrationof the composition to the patient.

In one embodiment, aminopyridine is administered to a subject at a doseand for a period sufficient to allow said subject to tolerate said dosewithout showing any adverse effects and thereafter increasing the doseat selected intervals of time until a therapeutic dose is achieved. Inone embodiment, the medicament is administered to a subject at a doseand for a period sufficient to allow said subject to tolerate said dosewithout showing any adverse effects and thereafter increasing the doseof aminopyridine at selected intervals of time until a therapeutic doseis achieved. For example, at the commencement of treatment aminopyridineis preferably administered at a dose less than 15 mg/day until atolerable state is reached. Suitably when said tolerable state isreached, the dose administered may be increased by amounts of at least5-15 mg/day until said therapeutic dose is reached.

Preferably, aminopyridine is administered at a dose of about 10-15 mgtwice daily (20-30 mg/day) depending upon the condition or symptomsbeing treated. The method can include scheduling administration of dosesof the pharmaceutical so that the concentration of the aminopyridine inthe patient is at about the minimum therapeutically effective level toameliorate the neurological condition, yet relatively lower compared tothe maximum concentration in order to enhance restful periods for thepatient during the day or night, depending on desired results or dosageschedule. Preferably the method provides for the treatment ofneurological diseases characterized by a degradation of nerve impulsetransmission comprising the step of administering to a patient acomposition of the present invention.

The formulations and compositions of the present invention exhibit aspecific, desired release profile that maximizes the therapeutic effectwhile minimizing adverse side effects. The desired release profile maybe described in terms of the maximum plasma concentration of the drug oractive agent (C_(max)) and the plasma concentration of the drug oractive agent at a specific dosing interval (Cτ). A ratio of C_(max) toC_(τ) (C_(max):C_(τ)) may be calculated from the observed C_(max) andC_(τ). A dosing interval (τ) is the time since the last administrationof the drug or active agent. In the present application, the dosinginterval (τ) is twelve (12) hours, therefore C_(τ) is the concentrationof the drug or active agent at twelve (12) hours from the lastadministration.

Additionally, the formulations and compositions of the present inventionexhibit a desired release profile that may be described in terms of themaximum plasma concentration of the drug or active agent at steady state(C_(maxSS)) and the minimum plasma concentration of the drug or activeagent at steady state (C_(minSS)). Steady state is observed when therate of administration (absorption) is equal to the rate of eliminationof the drug or active agent. A ratio of C_(maxSS) to C_(minSS)(C_(maxSS):C_(minSS)) may be calculated from the observed C_(maxSS) andC_(minSS). In addition, the formulations and compositions of the presentinvention exhibit a desired release profile that may be described interms of the average maximum plasma concentration of the drug or activeagent at steady state (C_(avSS)).

Another embodiment is a sustained release tablet of a sustained releasematrix and an aminopyridine, said tablet exhibits a release profile toobtain a C_(max):C_(τ) ratio in vivo of 1.0 to 3.5, and more preferablya C_(max):C_(τ) ratio of about 1.5 to about 3.0. In another preferredembodiment, the C_(max):C_(τ) ratio is about 2.0 to about 3.0. Theaminopyridine may comprise 4-aminopyridine. The sustained release matrixmay include for example, hydroxypropylmethylcellulose, or other ratecontrolling matrices that are suitable for controlling the release rateof an aminopyridine for use in the pharmaceutical compositions of thepresent invention.

Another embodiment is a sustained release tablet of a sustained releasematrix and an aminopyridine, said tablet exhibits a release profile toobtain a C_(max):C_(τ) ratio in vivo of 1.0 to 3.5 and a C_(avSS) ofabout 15 ng/ml to about 35 ng/ml, and more preferably a C_(max):C_(τ)ratio of about 1.5 to about 3.0. In another preferred embodiment, theC_(max):C_(τ) ratio is about 2.0 to about 3.0.

A further aspect is a sustained release composition comprising asustained release matrix and an aminopyridine, wherein said compositionprovides a C_(avss) of about 15 ng/ml to about 35 ng/ml. In a furtheraspect, a sustained release tablet comprising a sustained release matrixand an aminopyridine, said tablet exhibiting a C_(maxss) of about 20ng/ml to about 35 ng/ml is provided. The pharmacokinetic characteristicsof sustained release aminopyridine compositions and methods of treatingvarious neurological disorders are described in co-pendingPCT/US2004/008101 entitled “Stable Formulations of Aminopyrdines andUses Thereof” filed Apr. 17, 2004 and U.S. application Ser. No.11/010,828 entitled “Sustained Release Aminopyridine Composition” filedDec. 13, 2004, the contents of which are incorporated herein byreference in their entireties.

The amount of a pharmaceutically acceptable quality aminopyridine, salt,solvated, or prodrug thereof included in the pharmaceutical compositionof the present invention will vary, depending upon a variety of factors,including, for example, the specific potassium channel blocker used, thedesired dosage level, the type and amount of rate-controlling polymermatrix used, and the presence, types and amounts of additional materialsincluded in the composition. Preferably, the aminopyridine comprisesfrom about 0.1 to about 13% w/w, more preferably from about 0.5 to about6.25% w/w. In an even more preferable embodiment of the presentinvention the aminopyridine is present from about 0.5 to 4.75% w/w ofthe pharmaceutical composition. Accordingly, a weight percentage lessthan about 4.75% is desired. The amount of aminopyridine, or aderivative thereof, in the formulation varies depending on the desireddose for efficient drug delivery, the molecular weight, and the activityof the compound. The actual amount of the used drug can depend on thepatient's age, weight, sex, medical condition, disease or any othermedical criteria. The actual drug amount is determined according tointended medical use by techniques known in the art. The pharmaceuticaldosage formulated according to the invention may be administered once ormore times per day, preferably two or fewer times per day as determinedby the attending physician.

Suitable formulations and methods of manufacture are further describedin co-pending PCT/US2004/008101 entitled “Stable Formulations ofAminopyrdines and Uses Thereof” filed Apr. 17, 2004 and U.S. applicationSer. No. 11/010,828 entitled “Sustained Release AminopyridineComposition” filed Dec. 13, 2004, the contents of which are incorporatedherein by reference in their entireties.

The release matrix aminopyridine formulation is preferably fabricatedinto tablets, capsules or granules for oral use. The rate ofaminopyridine release from the tablets may be controlled by the erosionmechanism of the release matrix from which aminopyridine is released. Ingeneral, for producing a tablet on an industrial scale, the drug andpolymer are granulated alone or in combination. Preferably the releaseof the aminopyridine from the matrix of the pharmaceutical compositionis relatively linear over time. Preferably the matrix provides a releaseprofile that gives a therapeutically effective concentration of theaminopyridine in the plasma of the patient permitting a once per day ortwice per day dosing. Preferably the sustained release aminopyridineformulation for oral administration to patients includes from about0.0001 mole to about 0.0013 mole aminopyridine that provides a meanmaximum plasma concentration of aminopyridine from about 15 to about 180ng/ml, a mean T_(max) of about 2 to about 5 hours after administration,and a mean minimum plasma concentration of from about 10 to 60 ng/ml atabout 8-24 hours after administration.

The formulations of the invention are prepared by procedures known inthe art, such as, for example, by the dry or wet method. The methodselected for manufacturing affects the release characteristics of thefinished tablet. In one method, for example, the tablet is prepared bywet granulation in the presence of either water or an aqueous solutionof the hydrophilic polymer or using other binder as a granulating fluid.In alternative, organic solvent, such as isopropyl alcohol, ethanol andthe like, may be employed with or without water. The drug and polymermay be granulated alone or in combination. Another method forpreparation of the tablet which may be used requires using adrug-polymer dispersion in organic solvents in the presence or absenceof water. Where the aminopyridine or its derivative has very lowsolubility in water it may be advantageous to reduce the particle size,for example, by milling it into fine powder and in this way to controlthe release kinetics of the drug and enhance its solubility.

The hardness of the tablets of the present invention may vary, dependingon a variety of factors, including, for example, the relative amountsand specific types of ingredients used, the tableting equipmentemployed, and the selected processing parameters. The pressure used toprepare the tablets can influence the release profile of theaminopyridine into the patient. The pressure used to prepare the tabletsof the present invention may vary depending upon their surface area andthe amount and particle size of aminopyridine, additive, excipients, orbinders included in the tablet. The degree of hydration and solvation ofthe components in the composition will also be important in determiningthe hard ness of the tablets. Preferably the formed tablets have ahardness in the range of from 80-400 N, and more preferably from 150 to300 N.

The effects of various matrices, concentrations of aminopyridine, aswell as various excipients and additives to the composition on theconcentration of the channel blocker on the dissolution rate may bemonitored for example using a type H dissolution apparatus according toU.S. Pharmacopoeia XXII, or USP Apparatus II (Paddle Method). Clinicalevaluations may be used to study the effects on plasma levels of variousrelease matrices, concentrations of aminopyridine, as well as variousexcipients and additives. Plasma aminopyridine concentrations may beused to calculate pharmacokinetic data (release profiles) includingapparent absorption and elimination rates, area-under-the curve (AUC),maximum plasma concentration (C_(max)), time to maximum plasmaconcentration (T_(max)), absorption half-life (T_(1/2)(abs)), andelimination half-life (T_(1/2)(elim)). Pharmacodynamic effects may beassessed based upon response tests, such as muscle strength improvementor reduction in spasticity for patients with multiple sclerosis orspinal cord injury or other tests as would be known to those skilled inthe art. Plasma aminopyridine concentration in blood plasma or cerebralspinal fluid may be monitored using liquid chromatography/MS/MS assaymethods.

The drug delivery of the invention can utilize any suitable dosage unitform. Specific examples of the delivery system of the invention aretablets, tablets that disintegrate into granules, capsules, sustainedrelease microcapsules, spheroids, or any other means that allow for oraladministration. These forms may optionally be coated withpharmaceutically acceptable coating which allows the tablet or capsuleto disintegrates in various portions of the digestive system. Forexample a tablet may have an enteric coating that prevents it fromdissolving until it reaches the more basic environment of the smallintestine.

The dispersion of the aminopyridine throughout the release matriximparts enhanced stability characteristics in the dosage formulation.This enhanced stability is achieved without loss of the desiredsustained-release profile. Preferably the release profile, which may bemeasured by dissolution rate is linear or approximately linear,preferably the release profile is measured by the concentration of theaminopyridine in the plasma in the patient and is such to permit twicedaily (BID) dosing.

The pharmaceutical composition of the present invention can include alsoauxiliary agents or excipients, for example, glidants, dissolutionagents, surfactants, diluents, binders including low temperature meltingbinders, disintegrants, solubilizing agents and/or lubricants asdescribed in co-pending PCT/US2004/008101 entitled “Stable Formulationsof Aminopyrdines and Uses Thereof” filed Apr. 17, 2004 and U.S.application Ser. No. 11/010,828 entitled “Sustained ReleaseAminopyridine Composition” filed Dec. 13, 2004, the contents of whichare incorporated herein by reference in their entireties.

The active ingredient of the present invention may be mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient and in amounts suitable for use in the therapeuticmethods described herein. Various excipients may be homogeneously mixedwith the aminopyridines of the present invention as would be known tothose skilled in the art. For example, aminopyridines may be mixed orcombined with excipients such as but not limited to microcrystallinecellulose, colloidal silicon dioxide, lactose, starch, sorbitol,cyclodextrin and combinations of these.

To further improve the stability of the aminopyridine in the sustainedrelease composition, an antioxidant compound can be included. Suitableantioxidants include, for example: sodium metabisulfite; tocopherolssuch as α, β, δ-tocopherol esters and α.-tocopherol acetate; ascorbicacid or a pharmaceutically acceptable salt thereof; ascorbyl palmitate;alkyl gallates such as propyl gallate, Tenox PG, Tenox s-1; sulfites ora pharmaceutically acceptable salt thereof; BHA; BHT; andmonothioglycerol.

In another embodiment, the pharmaceutical composition of the presentinvention comprises a rate-controlling polymeric matrix comprising of ahydrogel matrix. For instance, an aminopyridine may be compressed into adosage formulation containing a rate-controlling polymer, such as HPMC,or mixture of polymers which, when wet, will swell to form a hydrogel.The rate of release of the aminopyridine from this dosage formulation issustained both by diffusion from the swollen tablet mass and by erosionof the tablet surface over time. The rate of release of theaminopyridine may be sustained both by the amount of polymer per tabletand by the inherent viscosities of the polymers used.

According to another aspect of the invention, there is provided astable, sustained-release oral dosage formulation which includes aneffective amount a aminopyridine dispersed in a release matrix, andwhich, upon administration to a patient or as part of a therapyregiment, provides a release profile (of therapeutically effective bloodplasma level of the aminopyridine) extending for a period of at least 6hours, preferably at least 12 hours. In another embodiment, the stable,controlled-release oral dosage form provides, upon administration to apatient, a therapeutically effective blood plasma level of theaminopyridine for a period of at least 6 hours, preferably at least 12hours, and more preferably at least 24 hours.

The dosage formulation may assume any form capable of delivering orallyto a patient a therapeutically effective amount of an aminopyridinedispersed in a rate-controlling polymer. Preferably, the dosageformulation comprises a monolithic tablet.

Tablet weight will also vary in accordance with, among other things, theaminopyridine dosage, the type and amount of rate-controlling polymerused, and the presence, types and amounts of additional materials.Assuming 4-aminopyridine dosages of from about 2 mg to about 120 mg;tablet weights can range from about 50 mg to about 1200 mg per tablet,and preferably from 250 to 500 mg, and more preferably about 400 mg.

The dosage formulation of the present invention may comprise also one ormore pharmaceutically acceptable excipients as mentioned above. Inpreferred embodiments, the dosage formulation will comprise diluents anda lubricant in addition to the aminopyridine unit dose and therate-controlling polymer. Particularly preferred diluents ismicrocrystalline cellulose sold under the name Avicel PH101, and aparticularly preferred lubricant is magnesium stearate. When thesematerials are used, the magnesium stearate component preferablycomprises from about 0.2 to about 0.75% w/w of the dosage formulation,and the microcrystalline cellulose along with the rate controllingpolymer and aminopyridine comprises the balance of the formulation. Forexample, a tablet formulation including a aminopyridine x % w/w, arate-controlling polymer y % w/w, and microcrystalline cellulose z %,the magnesium stearate amount would be (100−(x+y+z)) where0.2%≤(100−(x+y+z))≤0.75% w/w. As would be known to those skilled in theart, the amount of an additives such as magnesium stearate may varydepending upon the shear rate used to perform the mixing and the amountof such an additive may be changed without limitation to obtain asatisfactory dissolution rate or plasma level of the aminopyridine.

As used herein, the term “sustained-release” as it relates to theaminopyridine compositions includes the release of a aminopyridine fromthe dosage formulation at a sustained rate such that a therapeuticallybeneficial blood level below toxic levels of the aminopyridine ismaintained over a period of at least about 12 hours, preferably about 24hours or more. Preferably, the amount of the aminopyridine in the oraldosage formulations according to embodiments of the present inventionestablish a therapeutically useful plasma concentration through BIDadministration of the pharmaceutical composition.

If desired, the dosage formulations of this invention may be coated witha sustained-release polymer layer so as to provide additionalsustained-release properties. Suitable polymers that can be used to formthis sustained release layer include, for example, the release matriceslisted above. As desired, the dosage formulation of the invention can beprovided also with a light-protective and/or cosmetic film coating, forexample, film-formers, pigments, anti-adhesive agents and politicizes.Such a film-former may consist of fast-dissolving constituents, such aslow-viscosity hydroxypropylmethylcellulose, for example, Methocel E5 orD14, or Pharmacoat 606 (Shin-Etsu). The film coating may also containexcipients or enteric coatings customary in film-coating procedures,such as, for example, light-protective pigments, for example, ironoxide, or titanium dioxide, anti-adhesive agents, for example, talc, andalso suitable plasticizers such as, for example, PEG 400, PEG 6000,diethyl phthalate or triethyl citrate.

The compositions of the present invention may be used for the treatmentof neurological diseases characterized by a degradation of nerve impulsetransmission by administering to a patient the oral dosage formulationof the present invention. Preferably, the administration is twice dailydosage of a therapeutically effective amount of an aminopyridine, evenmore preferably, 4-AP dispersed in HPMC. The administration can alsoinclude scheduling administration of doses of the pharmaceutical so thatthe concentration of the aminopyridine in the patient is at about theminimum therapeutically effective level to ameliorate the neurologicalcondition, yet relatively low compared to the maximum concentration inorder to minimize side effects. The compositions may be administered toa subject at a dose and for a period sufficient to allow said subject totolerate said dose without showing any adverse effects and thereafterincreasing the dose of said active agent in the tablets at selectedintervals of time until a therapeutic dose is achieved in the subject.For example, at the commencement of treatment the active agent ispreferably administered at a dose less than about 15 mg/day until atolerable state is reached. The dose administered may then be increasedby amounts of at least 5-10 mg/day until a therapeutic dose is reached,preferably less than about 30 mg/day. For other diseases the amount ofthe aminopyridine required to reach a therapeutically effective amountfor treatment is described in U.S. Pat. No. 5,952,357 the contents ofwhich are incorporated herein by reference in their entirety.

Compositions of the present invention where the potassium channelblocker is a mono- or di-aminopyridine active agent are particularlysuitable for use in the treatment of a neurological disease that ischaracterized by demyelination of the central nervous system, moreespecially multiple sclerosis.

In one embodiment of the present invention, a method of treatingmultiple sclerosis is provided. Compositions of the present inventioncontaining a therapeutically effective amount of mono- ordi-aminopyridine active agent may be administered to a patient in needthereof. In particular, sustained release compositions comprising atleast about 5 milligrams of an aminopyridine, preferably 4-aminopyridinemay be administered at least once daily. In a preferred embodiment, asustained release composition containing from about 10 to about 15milligrams of 4-aminopyridine is administered twice daily. Treatment ofmultiple sclerosis may include increased walking speed, improved lowerextremity muscle strength or improved lower extremity muscle tone. Thesustained release aminopyridine composition is preferably administeredtwice daily. In certain embodiments, the composition may be administeredabout every 12 hours.

A further embodiment is a method of increasing walking speed in patientswith multiple sclerosis comprising administering to a patient at leastabout 5 milligrams of a sustained release aminopyridine composition,preferably at least about 10 to about 15 milligrams of a sustainedrelease aminopyridine composition.

A further embodiment is a method of increasing muscle tone or musclestrength in patients with multiple sclerosis comprising administering toa patient at least about 5 milligrams of a sustained releaseaminopyridine composition, preferably at least about 10 to about 15milligrams of a sustained release aminopyridine composition.

Fampridine is a potential therapy for MS with a unique mechanism ofaction. At concentrations of 1-2 μM or less, fampridine appears to be aspecific blocker of voltage dependent, neuronal potassium channels thataffect conduction in demyelinated axons. Fampridine has been shown torestore action potential conduction in damaged, poorly myelinated nervefibers, and it may also directly enhance synaptic transmission. Inprevious clinical trials, treatment with fampridine has been associatedwith a variety of neurological benefits in people with MS includingfaster walking and increased strength, as measured by standardneurological assessments.

Another aspect of the present invention provides for a method ofselecting individuals based on responsiveness to a treatment. In oneembodiment, the method comprises identifying a plurality of individuals;administering a test to each individual prior to a treatment period;administering a treatment, including, but not limited to administering atherapeutic agent or drug, to one or more of the individuals during thetreatment period; administering the test a plurality of times to eachindividual during the treatment period; and selecting one or moreindividuals, wherein the selected individuals exhibit an improvedperformance during a majority of the tests administered during thetreatment period as compared to the test administered prior to thetreatment period. In certain embodiments, the method may furthercomprise administering the test to each individual after the treatmentperiod, wherein the selected individuals further exhibit an improvedperformance during a majority of the tests administered during thetreatment period as compared to the test administered after thetreatment period.

It is important to note that this embodiment selects subjects who show apattern of change that is consistent with a treatment response, but doesnot define the full characteristics of that response. The criterionitself does not specify the amount of improvement nor does it specifythat the improvement must be stable over time. For example, aprogressive decline in effect during the course of the study period,even one resulting in speeds slower than the maximum non-treatmentvalue, would not be excluded by the criterion; as a specific example,changes from the maximum non-treatment value of, respectively, +20%,+5%, +1% and −30% during the double blind treatment period would qualifyas a response under the criterion, but would actually show a netnegative average change for the entire period, poor stability and anegative endpoint. Post-hoc analyses of studies discussed in greaterdetail below indicate that we may expect responders defined byconsistency of effect also to demonstrate increased magnitude andstability of benefit.

We have found this embodiment particularly applicable in our analysis offampridine in patients suffering from multiple sclerosis. Clinicians whoregularly prescribe compounded fampridine for MS have reported that onlya proportion of their patients appear to respond with clear clinicalbenefits, and that, in their judgment, this proportion may be around onethird. This extent of responsiveness may be related to the proposedmechanism of action, which is the restoration of conduction indemyelinated axons via the blockade of voltage-dependent potassiumchannels. Only a proportion of MS patients would be expected to possessaxons of appropriate functional relevance that are susceptible to thesedrug effects, given the highly variable pathology of the disease.Currently, there is insufficient understanding of the disease to allowfor pre-trial selection of potentially responsive patients. However, theexistence of a subset of patients who respond consistently to the drugcan be supported by quantitative observations in our own clinicalstudies discussed below.

Before treatment, the subjects in these two trials exhibited averagewalking speeds on the TW25 measure of approximately 2 feet per second(ft/sec). This is a significant deficit, since the expected walkingspeed for an unaffected individual is 5-6 ft/sec. Subjects in MS-F202were selected for TW-25 walking time at screening of 8-60, which isequivalent to a range in speed of 0.42-3.1 ft/sec. Variability offunctional status is an inherent characteristic of MS, and this can beseen in repeated measurement of walking speed over the course of weeksor months. At any of the three visits during the stable treatmentperiod, 15-20% of placebo-treated subjects showed >20% improvement frombaseline walking speed, a threshold chosen as one that indicates a truechange in walking speed over background fluctuations. A largerproportion of the Fampridine-SR treated subjects showed suchimprovements, but this difference was not statistically significant,given the sample size and placebo response rate.

Given the often large variations in function experienced by people withMS, it is difficult for the subject or a trained observer to separate atreatment-related improvement from a disease-related improvement withoutthe element of consistency over time. Consistency of benefit mighttherefore be expected to be a more selective measure of true treatmenteffect than magnitude of change. Based on this rationale, the responsesof the individual subjects in the MS-F202 trial were examined for thedegree to which their walking speed showed improvement during thedouble-blind treatment period and returned towards pre-treatment valuesafter they were taken off drug, at follow-up. This subject-by-subjectexamination yielded a subgroup of subjects whose pattern of walkingspeed over time appeared to be consistent with a drug response. This ledto the analysis illustrated in FIG. 1. This compares the placebo andFampridine-SR treated groups with respect to the number of visits duringthe double-blind treatment period in which walking speed on the TW25 wasfaster than the maximum speed out of all five of the non-treatmentvisits (four visits prior to randomization and one follow-up visit afterthe drug treatment period).

The placebo-treated group showed a clear pattern of exponential declinein numbers of subjects with higher numbers of “positive” visits. This iswhat would be expected from a random process of variability. Incontrast, the pattern of response in the Fampridine-SR treated groupstrongly diverged from this distribution; much larger numbers ofFampridine-SR treated subjects showed three or four visits with higherwalking speeds than the maximum speed of all five non-treatment visitsand less than half of the expected proportion had no visits with higherspeeds. These results indicate that there was a sub-population ofsubjects in the Fampridine-SR treated group that experienced aconsistent increase in walking speed related to treatment.

This analysis suggests that a relatively highly selective criterion fora likely treatment responder would be: a subject with a faster walkingspeed for at least three (i.e., three or four) of the four visits duringthe double blind treatment period compared to the maximum value for allfive of the non-treatment visits. The four visits before initiation ofdouble-blind treatment provide an initial baseline against which tomeasure the consistency of response during the four treatment visits.The inclusion of the follow-up visit as an additional component of thecomparison was found valuable primarily in excluding those subjects whodid not show the expected loss of improvement after coming off the drug.These are likely to be subjects who happened by chance to have improvedin their MS symptoms around the time of treatment initiation, but whoseimprovement did not reverse on drug discontinuation because it wasactually unrelated to drug. Thus, incorporating the follow-up visit aspart of the criterion may help to exclude false positives, if the TW25speed remains high at follow-up.

As described in Example 5, below, this responder criterion was met by8.5%, 35.3%, 36.0%, and 38.6% of the subjects in the placebo, 10 mg, 15mg, and 20 mg b.i.d. treatment groups, respectively, showing a highlysignificant and consistent difference between placebo and drug treatmentgroups. Given that there was little difference in responsiveness betweenthe three doses examined, more detailed analyses were performedcomparing the pooled Fampridine-SR treated groups against theplacebo-treated group. The full results of this analysis for study aredescribed in the following sections. These show that the responder groupso identified experienced a >25% average increase in walking speed overthe treatment period and that this increase did not diminish across thetreatment period. The responder group also showed an increase in SubjectGlobal Impression score and an improvement in score on the MSWS-12.

Additional features and embodiments of the present invention areillustrated by the following non-limiting examples.

Example 1

This example illustrates preparation of compositions of the presentinvention and their release of an aminopyridine. Tablets in accordancewith the present invention having dosages of 5 mg, 7.5 mg and 12.5 mgrespectively were manufactured at 5 Kg scale. Materials were used in theamounts shown in Table 1.

TABLE 1 % w/w % w/w % w/w Milled 4-AP 1.25 1.875 3.125 (#50 mesh)Methocel K100LV 60 60 60 Avicel PH101 38.15 37.525 36.275 Magnesiumstearate 0.2 0.2 0.2 Aerosil 200 0.4 0.4 0.4 Equipment Tablet Horn Noakequipped with 13 × 8 mm oval tooling Press press speed 42,000 tablets/hrTablet Weight Range 386-404 388-410 388-406 (mg) (96.5-101.0%)(97.0-102.5%) (97.0-101.5%) Tablet Hardness Range 200-262 179-292150-268 (N) Tablet Potency - 97.1 99.1 100.2 mg/tab. (% LC) Mean CU(mg/tab.)/ 5.0 mg/1.0% 7.4 mg/0.7% 12.4 mg/1.1% % CV CU Discrete Samples5.0 mg/1.2% 7.5 mg/1.8% 12.3/1.1% (mg/tab.)/% CV Dissolution (%/hr) Mean(SD) Mean (SD) Mean (SD) 1 28.9 1.1 29.2 1.8 25.9 1.1 2 42.7 1.8 42.11.6 40.2 2.5 3 52.8 1.4 53.0 1.0 49.8 2.1 4 61.4 2.2 61.8 1.5 60.1 2.4 675.7 3.1 75.2 1.6 74.8 2.7 10  95.5 3.3 98.7 1.4 93.2 0.9

Prior to blending, 4-AP was milled through #50 mesh screen using aFitzmill® comminutor. The materials were added into a Gral 25 bowl inthe following order: half Methocel K100LV, Avicel PH101, Aerosil 200,milled 4-AP and the remaining Methocel K100LV. The mix was blended for15 minutes at 175 rpm, then the magnesium stearate was added and wasfurther blended for 5 minutes at 100 rpm. Samples were taken from topand bottom positions for blend potency analysis. Weight and hardnesschecks were performed every 15 minutes by the check-master E3049.Discrete tablet samples were taken during the compression process toevaluate intra batch content uniformity.

Example 2

This example illustrates that the pharmacokinetic profile of fampridinein compositions of the present invention is altered by administration ina sustained release tablet matrix compared to immediate release andcontrolled release formulations.

There is a delay in absorption manifested by a lower peak concentration,without any effect on the extent of absorption. When given as a single12.5 mg dose, the peak concentration is approximately two-thirds loweras compared to peak values following administration of the IRformulation; the time to reach peak plasma levels was delayed by about 2hours. As with the IR formulation, food delayed the absorption ofFampridine-SR. The absorption of fampridine was approximately 50% slowerfollowing ingestion of a fatty meal, although due to the flatness of theabsorption curve, this may be exaggerated value. Extent of absorptiondid not differ, as values for Cmax and AUC were comparable as summarizedin Table 2.

TABLE 2 Pharmacokinetic Parameter Values (Mean ± SD) in Studies UsingFampridine SR, CR, and IR Formulations: Single Dose Studies in HealthyAdult Male Volunteers C_(MAX) AUC (0-∞) Study Number Dose (mg)Fed/Fasted (ng/mL) t_(MAX) (hours) (ng hr/mL) 0494006 12.5 SR Fed 28.7 ±4.3 5.3 ± 0.8 257.0 ± 62.7 N = 12 (PD12265) Fasted 25.6 ± 3.8 2.8 ± 1.3269.9 ± 44.4 12.5 IR Fasted  79.3 ± 16.3 0.9 ± 0.4 294.2 ± 55.6(PD12266) 1194002 12.5 SR Fasted 28.5 ± 4.3 2.9 ± 2.4 285.9 ± 37.8 N =12 (PD12907) 12.5 CR Fasted 37.7 ± 9.9 3.6 ± 0.9 300.0 ± 53.6 (4n806)12.5 IR Fasted  83.5 ± 23.5 0.79 ± 0.3  274.0 ± 59.2 (PS644)

Example 3

This example details the pharmacokinetic properties of Fampridine-SR intablets of the present invention administered to patients with multiplesclerosis. Plasma samples were analyzed for fampridine using a validatedLC/MS/MS assay with a sensitivity of 2 ng/mL. Noncompartmentalpharmacokinetic parameter values were calculated using standardmethodology.

This was an open-label, multi-center, dose proportionality study oforally administered fampridine in patients with multiple sclerosis.Single doses of fampridine were to be given in escalating doses (5 mg,10 mg, 15 mg, and 20 mg) with at least a four-day interval betweenadministration of each dose of drug. Safety evaluations were to beperformed during the 24 hour period following administration offampridine and blood samples were to be taken at the following times todetermine pharmacokinetic parameters; hour 0 (pre-dose), hours 1-8, andhours 10, 12, 14, 18, and 24.

Twenty-three subjects received all 4 treatments, and one subjectreceived only 3 treatments; data from all treatments were analyzed.Dose-dependent parameters (e.g., peak plasma concentration andareas-under-the curve) were normalized to a 10 mg dose for among-dosecomparisons. Overall observed time of the peak plasma concentration(mean and its 95% confidence interval) was 3.75 (3.52, 3.98) h, observedpeak plasma fampridine concentration (normalized to a 10 mg dose) was24.12 (23.8, 26.6) ng/ml, area-under-the-concentration-time curve(normalized to a 10 mg dose) was estimated to be 254 (238, 270) ng·h/ml,extrapolated area-under-the-concentration-time curve (normalized to a 10mg dose) was 284 (266, 302) ng·h/ml, terminal rate constant equaled 0.14(0.13, 0.15) h⁻¹, terminal half-life was 5.47 (5.05, 5.89) h andclearance divided by bioavailability (CL/F) was equal to 637 (600, 674)ml/min.

Dizziness was the most common treatment-related adverse event. Othertreatment related adverse events included amblyopia, asthenia, headache,and ataxia. There were no clinically significant changes in clinicallaboratory values, ECG parameters, vital signs, physical examinationfindings, or neurological examination findings noted over the course ofthis study.

When the plasma concentrations of fampridine were normalized to the 10.0mg dose levels, there were no significant differences between anypharmacokinetic parameter (AUC, C_(max), t_(1/2)) in the 5-20 mg doserange. Fampridine was well tolerated at the doses used in this study.Dose-normalized (to a 10 mg dose) pharmacokinetic parameter values aresummarized in Table 3.

TABLE 3 Dose-Normalized (at 10 mg) Pharmacokinetic Parameter Values(Mean ± SEM) Following Single Oral Administration of Fampridine-SR toPatients with MS. Dose C_(MAX)-norm t_(MAX) AUC-norm t_(1/2) Cl/F (mg)(ng/mL) (hours) (ng hr/mL) (hours) (mL/min)  5 26.2 ± 0.6 3.9 ± 0.2244.2 ± 9.4 5.8 ± 0.5 619.8 ± 36.2 (n = 24) 10 25.2 ± 0.7 3.9 ± 0.3252.2 ± 7.8 5.6 ± 0.4 641.4 ± 39.1 (n = 24) 15 24.6 ± 0.7 3.6 ± 0.3263.0 ± 7.4 5.5 ± 0.4 632.4 ± 39.0 (n = 24) 20 24.6 ± 0.8 3.6 ± 0.3255.6 ± 6.9 5.1 ± 0.3 653.9 ± 37.1 (n = 23)

Example 4

This example describes the results of an open-label study to assess thesteady state pharmacokinetics of orally administered fampridine(4-aminopyridine) compositions of the present invention in subjects withMultiple Sclerosis. This study was an open-label multiple dose study ofFampridine-SR intended to assess steady state pharmacokinetics in 20patients with MS who previously completed the study summarized in Table4. Fampridine-SR (40 mg/day) was administered as two 20 mg doses, givenas one morning and one evening dose for 13 consecutive days, with asingle administration of 20 mg on Day 14. Blood samples forpharmacokinetic analysis were collected on Days 1, 7/8, and 14/15 at thefollowing intervals: immediately prior to drug administration(baseline), hourly for the first 8 hours, and 10, 12, and 24 hourspost-dose. Additional blood samples were collected 14, 18, and 20 hourspost-dose on Day 14, and 30 and 36 hours post-dose on Day 15.

Pharmacokinetic parameter estimates following the first dose in thesepatients in this study on Day 1 were comparable to those determined whenthey participated in the study summarized in Table 4. No significantdifference in T_(max) was detected among the four means (Singledose=3.76 h; Day 1=3.78 h; Day 8=3.33 h; Day 15=3.25 h). C_(max) andC_(max)/C_(τ) on Days 8 (C_(max)=66.7 ng/ml) and 15 (C_(max)=62.6 ng/ml)were significantly greater than those of the single dose treatment andof Day 1 (C_(max)=48.6 ng/ml), reflecting accumulation of the drug withmultiple dosing.

There was no significant difference among the four occasions with regardto either T or C and no difference in C_(max), C_(max)/C_(τ), CL/F orAUC_(0-τ) between Days 8 and 15. Further AUC on Days 8 and 15 did notdiffer significantly from total AUC with single dose treatment.Likewise, the estimates of CL/F on Days 8 and 15 and of λ and T_(1/2) onDay 15 did not differ significantly from those with single dose.

Steady-state was attained by Day 7/8 as evidence by the lack ofdifferences in C_(max) or AUC between Days 7/8 and 14/15; there was noapparent unexpected accumulation. Likewise, the estimates of Cl/F onDays 7/8 and 14/15 of and of T_(1/2) on Day 14/15 did not differsignificantly from those given a single dose. On the final day ofdosing, mean C_(max) was 62.6 ng/mL, occurring 3.3 hours post-dose. TheT_(1/2) was 5.8 hours. These values are similar to those observed inpatients with chronic SCI receiving similar doses of this formulation.These results are summarized in Table 4.

TABLE 4 Pharmacokinetic Parameter Values (Mean and 95% CI) FollowingMultiple Oral Doses of Fampridine-SR (40 mg/day) to 20 Patients with MS.Parameter C_(MAX) t_(MAX) AUC₍₀₋₁₂₎ t_(1/2) Cl/F Day (ng/mL) (hours) (nghr/mL) (hours) (mL/min) Day 1 48.6 3.8 NE NE NE (42.0, 55.3) (3.2, 4.3)Day 7/8 66.7 3.3 531 NE 700 (57.5, 76.0) (2.8, 3.9) (452, 610) (557,884) Day 62.6 3.3 499 5.8 703 14/15 (55.7, 69.4) (2.6, 3.9) (446, 552)(5.0, 6.6) (621, 786)

Dizziness was the most common treatment-related adverse event. Othertreatment-related adverse events that occurred included nausea, ataxia,insomnia, and tremor. There were no clinically significant changes inmean clinical laboratory values, vital signs, or physical examinationfindings from baseline to last visit. There were no apparent clinicallysignificant changes in corrected QT intervals or QRS amplitudes afteradministration of fampridine.

Fampridine was well tolerated in subjects with multiple sclerosis whoreceive twice daily doses (20 mg/dose) of fampridine for two weeks. Asignificant increase was observed in C_(max), and C_(max)/C_(τ) on Days8 and 15 relative to those on Day 1 and with single dose treatment,reflecting accumulation of fampridine with multiple dosing. A lack ofsignificant differences in C_(max), C_(max)/C_(τ), CL/F or AUC_(0-τ)between Days 8 and 15 suggest that near steady-state is reached by Day8. There was no evidence of significant changes in pharmacokineticsduring a two-week period of multiple dosing with fampridine.

Example 5

This example provides an embodiment of a method of treating subjectswith a sustained release fampridine formulation and a responder analysisof the present invention. This was a Phase 2, double-blind,placebo-controlled, parallel group, 20-week treatment study in 206subjects diagnosed with Multiple Sclerosis. This study was designed toinvestigate the safety and efficacy of three dose levels ofFampridine-SR, 10 mg b.i.d., 15 mg b.i.d., and 20 mg b.i.d. in subjectswith clinically definite MS. The primary efficacy endpoint was anincrease, relative to baseline, in walking speed, on the Timed 25 FootWalk. Secondary efficacy measurements included lower extremity manualmuscle testing in four groups of lower extremity muscles (hip flexors,knee flexors, knee extensors, and ankle dorsiflexors); the 9-Hole PegTest and Paced Auditory Serial Addition Test (PASAT 3″); the Ashworthscore for spasticity; Spasm Frequency/Severity scores; as well as aClinician's (CGI) and Subject's (SGI) Global Impressions, a Subject'sGlobal Impression (SGI), the Multiple Sclerosis Quality of LifeInventory (MSQLI) and the 12-Item MS Walking Scale (MSWS-12).

At the first visit (Visit 0) subjects were to enter into a two-weeksingle-blind placebo run-in period for the purpose of establishingbaseline levels of function. At Visit 2 subjects were to be randomizedto one of four treatment groups (Placebo or Fampridine-SR 10 mg, 15 mg,20 mg) and begin two weeks of double-blind dose-escalation in the activedrug treatment groups (B, C and D). Group A were to receive placebothroughout the study. Subjects in the 10 mg (Group B) arm of the studytook a dose of 10 mg approximately every 12 hours during both weeks ofthe escalation phase. The 15 mg (Group C) and 20 mg (Group D) dosesubjects took a dose of 10 mg approximately every 12 hours during thefirst week of the escalation phase and titrated up to 15 mg b.i.d. inthe second week. Subjects were to be instructed to adhere to an “every12 hour” dosing schedule. Each subject was advised to take themedication at approximately the same time each day throughout the study;however, different subjects were on differing medication schedules(e.g., 7 AM and 7 PM; or 9 AM and 9 PM). After two weeks, the subjectswere to return to the clinic at Visit 3 for the start of the stable dosetreatment period. The first dose of the double-blind treatment phase atthe final target dose (placebo b.i.d. for the Group A, 10 mg b.i.d. forGroup B, 15 mg b.i.d. for Group C, and 20 mg b.i.d. for Group D) wastaken in the evening following Study Visit 4. Subjects were to beassessed five times during the 12-week treatment period. Following the12-week treatment phase there was to be a one-week down titrationstarting at Visit 9. During this down-titration period, group B was toremain stable at 10 mg b.i.d. and Group C was to be titrated to 10 mgb.i.d., while group D was to have a change in the level of dose duringthe week (15 mg b.i.d. for the first three days and 10 mg b.i.d. for thelast four days). At the end of the down titration period at Visit 10,subjects were to enter a two-week washout period where they did notreceive any study medication. The last visit (Visit 11) was to bescheduled two weeks after the last dosing day (end of the downwardtitration). Plasma samples were collected at each study site visit otherthan Study Visit 0.

The primary measure of efficacy was improvement in average walkingspeed, relative to the baseline period (placebo run-in), using the Timed25 Foot Walk from the Multiple Sclerosis Functional Composite Score(MSFC). This is a quantitative measure of lower extremity function.Subjects were instructed to use whatever ambulation aids they normallyuse and to walk as quickly as they could from one end to the other endof a clearly marked 25-foot course. Other efficacy measures included theLEMMT, to estimate muscle strength bilaterally in four groups ofmuscles: hip flexors, knee flexors, knee extensors, and ankledorsiflexors. The test was performed at the Screening Visit and at StudyVisits 1, 2, 4, 7, 8, 9 and 11. The strength of each muscle group wasrated on the modified BMRC scale: 5=Normal muscle strength;4.5=Voluntary movement against major resistance applied by the examiner,but not normal; 4=Voluntary movement against moderate resistance appliedby the examiner; 3.5=Voluntary movement against mild resistance appliedby the examiner; 3=Voluntary movement against gravity but notresistance; 2=Voluntary movement present but not able to overcomegravity; 1=Visible or palpable contraction of muscle but without limbmovement; and 0=Absence of any voluntary contraction. Spasticity in eachsubject was assessed using the Ashworth Spasticity Score. The AshworthSpasticity Exam was performed and recorded at the Screening Visit and atStudy Visits 1, 2, 4, 7, 8, 9 and 11.

Protocol Specified Responder Analysis. To supplement the primaryanalysis, a categorical “responder” analysis was also conducted.Successful response was defined for each subject as improvement inwalking speed (percent change from baseline) of at least 20%. Subjectswho dropped out prior to the stable dose period were considerednon-responders. The proportions of protocol specified responders werecompared among treatment groups using the Cochran-Mantel-Haenszel test,controlling for center.

Post hoc analysis of this study suggested that a relatively highlyselective criterion for a likely treatment responder would be a subjectwith a faster walking speed for at least three visits during the doubleblind treatment period as compared to the maximum value among a set offive non-treatment visits (four before treatment and one afterdiscontinuation of treatment). The four visits before initiation ofdouble-blind treatment provided an initial baseline against which tomeasure the consistency of response during the four double-blindtreatment visits. The inclusion of the follow-up visit as an additionalcomponent of the comparison was useful primarily in excluding thosesubjects who may be false positives, i.e., did not show the expectedloss of improvement after coming off the drug. Treatment differences inthe proportion of theses post hoc responders were analyzed using theCochran-Mantel-Haenszel (CMH) test, controlling for center.

To validate the clinical meaningfulness of the post hoc respondervariable, (post hoc) responders were compared against the (post hoc)non-responders, on the subjective variables: (i) Change from baseline inMSWS-12 over the double-blind; (ii) SGI over the double-blind; and (iii)Change from baseline in the CGI over the double-blind; to determine ifsubjects with consistently improved walking speeds during thedouble-blind could perceive improvement relative to those subjects whodid not have consistently improved walking speeds. For the subjectivevariables, differences between responder status classification(responder or non-responder) were compared using an ANOVA model witheffects for responder status and center.

Results. A total of 206 subjects were randomized into the study: 47 wereassigned to placebo, 52 to 10 mg bid Fampridine-SR (10 mg bid), 50 to 15mg bid Fampridine-SR (15 mg bid), and 57 to 20 mg bid Fampridine-SR (20mg bid). The disposition of subjects is presented in Table 5 below.

TABLE 5 Summary of subject disposition (all randomized population)Treatment Group: N (%) Placebo 10 mg bid 15 mg bid 20 mg bid TotalSubjects Randomized 47 52 50 57 206 Took at Least One Dose 47 (100%) 52(100%)  50 (100%) 57 (100%) 206 (100%)  (Included in Safety Analysis)ITT Population 47 (100%) 51 (98.1%) 50 (100%) 57 (100%) 205 (99.5%)Discontinued Subjects  2 (4.3%) 2 (3.8%)  1 (2.0%)   6 (10.5%) 11 (5.3%)Note: Percentages are based on the number of randomized subjects.

All 206 randomized subjects took at least one dose of study medicationand were included in the safety population. One subject (subject#010/0710 mg bid group) was excluded from the ITT population (lost to follow-upafter 8 days of placebo run-in). A total of 11 subjects discontinuedfrom the study.

The population consisted of 63.6% females and 36.4% males. The majorityof the subjects were Caucasian (92.2%), followed by Black (4.9%),Hispanic (1.5%), those classified as ‘Other’ (1.0%), and Asian/PacificIslander (0.5%). The mean age, weight, and height of the subjects were49.8 years (range: 28-69 years), 74.44 kilograms (range: 41.4-145.5kilograms), and 168.84 centimeters (range: 137.2-200.7 centimeters),respectively. Most of the subjects (52.4%) had a diagnosis type ofsecondary progressive with about equal amounts of relapsing remitting(22.8%) and primary progressive (24.8%) subjects. The mean duration ofdisease was 12.00 years (range: 0.1-37.5 years) while the mean ExpandedDisability Status Scale (EDSS) at screening was 5.77 units (range:2.5-6.5 units). The treatment groups were comparable with respect to allbaseline demographic and disease characteristic variables.

Results for the key efficacy variables at baseline for the ITTpopulation are further summarized in Table 6 below.

TABLE 6 Summary of key efficacy variables at baseline (ITT population)Treatment Group: Mean (SD) placebo 10 mg bid 15 mg bid 20 mg bidTreatment. Parameter N = 47 N = 51 N = 50 N = 57 p-value Walking Speed(ft/sec) 1.87 (0.902) 1.94 (0.874) 1.99 (0.877) 2.04 (0.811) 0.752 LEMMT4.05 (0.690) 3.98 (0.661) 4.00 (0.737) 3.98 (0.634) 0.964 SGI 4.38(0.795)  4.32 (0.999)* 4.56 (1.110) 4.25 (0.969) 0.413 MSWS-12 75.71(16.566) 76.31 (16.186) 74.60 (17.671) 76.83 (18.124) 0.923 *One subjectdid not have a baseline value.

With respect to the 205 subjects in the ITT population, mean values forbaseline walking speed, LEEMT, SGI, and MSWS-12 were approximately 2feet per second, 4 units, 4.5 units, and 76 units, respectively. Thetreatment groups were comparable with respect to these variables as wellas all the other efficacy variables at baseline.

Descriptive statistics for the average walking speed (ft/sec) by studyday based on the Timed 25-Foot Walk are presented in Table 7 and FIG. 2.The timed 25 foot walk showed a trend toward increased speed during thestable dose period for all three dose groups, though the averageimprovement declined during the treatment period.

TABLE 7 Average walking speeds (ft/sec) by study day (observed cases,ITT population) Summary Statistics Over Time Study day Treat- follow-ment base titration 1st stbl 2nd stbl 3rd stbl up placebo Mean 1.87 1.891.90 1.89 1.89 1.86 (SD) (0.902) (0.876) (0.908) (0.891) (0.914) (0.933)N# 47 47 46 46 45 45 10 mg Mean 1.94 2.20 2.09 2.12 2.00 1.88 bid (SD)(0.874) (0.979) (0.955) (1.043) (1.016) (0.970) N 51 51 51 51 50 48 15mg Mean 1.99 2.25 2.16 2.14 2.18 1.83 bid (SD) (0.877) (0.995) (0.986)(0.957) (0.932) (0.952) N 50 49 49 48 48 47 20 mg Mean 2.04 2.26 2.222.19 2.04 1.83 bid (SD) (0.811) (0.936) (0.893) (0.936) (0.996) (0.822)N 57 55 52 51 49 55 #The treatment sample sizes presented in the figurelegend represent the number of ITT subjects. Sample sizes at individualtime points may be smaller than those in the ITT population due todropouts or missed assessments.

During double-blind treatment, all the Fampridine-SR groups exhibitedmean walking speeds between 2.00 and 2.26 feet per second, while themean value in the placebo group was consistently about 1.90 feet persecond. It should be noted that, at the third stable-dose visit, boththe 10 mg bid and 20 mg bid group means dropped-off from what would beexpected under the assumption that treatment benefit is consistent overtime. This may or may not have been due to chance; further studiesshould provide additional evidence for either case. After double-blindmedication was discontinued, all the treatment groups converged toapproximately the same mean value at follow-up.

Results for the primary efficacy variable (percent change in averagewalking speed during the 12-week stable dose period relative to baselinebased on the 25-foot walk) are summarized in FIG. 3. The timed 25 footwalk showed a trend toward increased speed during the stable dose periodfor all three dose groups, though the average improvement declinedduring the treatment period, as shown in FIG. 3. The mean percentchanges in average walking speed during the 12-week stable dose period(based on adjusted geometric mean change of the log-transformed walkingspeeds) were 2.5%, 5.5%, 8.4%, and 5.8% for the placebo, 10 mg bid, 15mg bid, and 20 mg bid groups, respectively. There were no statisticaldifferences between any Fampridine-SR groups and the placebo group.

Results for the protocol specified responder analysis (subjects withaverage changes in walking speed during the 12 weeks of stabledouble-blind treatment of at least 20%) are summarized in FIG. 4. Thepercentages of subjects with average changes in walking speed during the12-week stable dose period of at least 20% (pre-defined responders) were12.8%, 23.5%, 26.5%, and 16.1% for the placebo, 10 mg bid, 15 mg bid,and 20 mg bid groups, respectively. There were no statisticallysignificant differences between any of the Fampridine-SR groups and theplacebo group.

Descriptive statistics for the average overall Lower Extremity ManualMuscle Testing (LEMMT) by study day are presented in Table 8 and in FIG.5.

TABLE 8 Average overall LEMMT by Study Day Summary Statistics Over TimeStudy day Treat- follow- ment base titration 1st stbl 2nd stbl 3rd stblup placebo Mean 4.05 4.00 4.02 4.03 4.00 4.02 (SD) (0.690) (0.705)(0.687) (0.696) (0.679) (0.738) N# 47 46 46 46 45 45 10 mg Mean 3.984.09 4.06 4.09 4.07 3.89 bid (SD) (0.661) (0.641) (0.650) (0.685)(0.642) (0.631) N 51 50 51 51 50 49 15 mg Mean 4.00 4.16 4.11 4.09 4.174.08 bid (SD) (0.737) (0.653) (0.645) (0.659) (0.618) (0.674) N 50 49 4949 49 46 20 mg Mean 3.98 4.08 4.03 3.98 4.07 3.92 bid (SD) (0.634)(0.639) (0.659) (0.714) (0.649) (0.650) N 57 54 52 52 48 55 #Thetreatment sample sizes presented at individual time points may besmaller than those in the ITT population due to dropouts or missedassessments.

During double-blind treatment, all the Fampridine-SR groups exhibited anumerical pattern of larger mean LEMMT scores than placebo (except the20 mg bid group at the 2^(nd) stable dose visit). After double-blindmedication was discontinued, with the exception of the 15 mg bid group,all the group means were lower than they were at baseline.

Results for the average change in LEMMT during the 12-week stable doseperiod relative to baseline are summarized in FIG. 6. The mean changesin overall LEMMT during the 12-week stable dose period were −0.05 units,0.10 units, 0.13 units, and 0.05 units for the placebo, 10 mg bid, 15 mgbid, and 20 mg bid groups, respectively. Improvements in LEMMT weresignificantly greater in the 10 mg bid and 15 mg bid groups compared tothe placebo group; there was no significant difference between the 20 mgbid group and the placebo group.

No statistically significant differences were detected among treatmentgroup based on any of the other secondary efficacy variables, as shownin Table 9.

TABLE 9 Changes in secondary efficacy variables from baseline during the12-week stable dose period Treatment Group placebo 10 mg bid 15 mg bid20 mg bid Parameter N = 47 N = 51 N = 50 N = 57 Ashworth Score N 46 5149 53 Mean (SD) −0.11 (0.377)  −0.04 (0.449)  −0.06 (0.375)  0.02(0.466) p-value (each dose vs. placebo) 0.802 0.826 0.275 CGI N 45 50 4952 Mean (SD) 0.0 (0.66) −0.2 (0.72)  −0.1 (0.85)  0.0 (0.78) p-value(each dose vs. placebo) 0.772 0.997 0.996 SGI N 46 50 49 53 Mean (SD)−0.2 (0.96)  0.0 (1.27) −0.1 (1.11)  −0.1 (0.86)  p-value (each dose vs.placebo) 0.704 0.953 0.968 PASAT N 46 51 49 53 Mean (SD) 2.17 (4.016)2.13 (3.394) 0.90 (3.274) 0.65 (4.590) p-value (each dose vs.placebo) >0.999 0.306 0.218 MSFC N 46 51 49 52 Mean (SD) 0.08 (0.205)0.10 (0.310) 0.90 (0.224) 0.06 (0.194) p-value (each dose vs. placebo)0.977 >0.999 0.968 MSWS-12 N 46 51 49 52 Mean (SD) −3.56 (14.548) −5.53(16.154) −7.32 (16.295) −5.76 (15.296) p-value (each dose vs. placebo)0.718 0.445 0.617 Note: The treatment sample sizes presented in thetreatment heading represent the number of ITT subjects. Sample sizes forindividual variables may be smaller due to dropouts or missedassessments. Note: For each variable, the p-values (versus placebo) areDunnett-adjusted.

While pre-planned analyses of the primary efficacy endpoint providedinsufficient evidence of treatment benefits for any of the Fampridine-SRdoses, subsequent analysis revealed the existence of a subset ofsubjects who responded to the drug with clinical meaningfulness. Thesesubjects exhibited walking speeds while on drug that were consistentlybetter than the fastest walking speeds measured when the subjects werenot taking active drug.

The post hoc responder rates based on consistency of improved walkingspeeds were significantly higher in all three active dose groups (35, 36and 39%) compared to placebo (9%; p<0.006 for each dose group, adjustingfor multiple comparisons) as shown in FIG. 7.

Given that there was little difference in responsiveness between thethree doses examined, more detailed analyses were performed comparingthe pooled Fampridine-SR treated groups against the placebo-treatedgroup. FIG. 8 summarizes, for the placebo and the pooled Fampridine-SRgroup, the percentage of post hoc responders. The number of subjects whomet he post hoc responder criterion in the pooled Fampridine-SR treatedgroup was 58 (36.7%) compared to 4 (8.5%) in the placebo-treated group,and this difference was statistically significant (p<0.001).

To validate the clinical meaningfulness of the post hoc respondervariable, the 62 responders (58 fampridine and 4 placebo) were comparedagainst the 143 non-responders (100 fampridine and 43 placebo) on thesubjective variables to determine if subjects with consistently improvedwalking speeds during the double-blind could perceived benefit relativeto those subjects who did not have consistently improved walking speeds.The results are summarized in FIG. 9 and indicate that consistency inwalking speed had clinical meaningfulness for the subjects in this studysince the responders had (over the double-blind period) significantlybetter changes from baseline in MSWS-12 and significantly bettersubjective global scores. In addition, the responders were ratedmarginally better than the non-responders by the clinicians during thedouble-blind. Thus, responders experienced clinically meaningfulimprovements in their MS symptoms, and treatment with fampridinesignificantly increased the chances of such a response.

To establish baseline comparability among the responder analysis groups,analyses were performed on the baseline demographic variables, keyneurological characteristics and the relevant efficacy variables atbaseline. In general, the responder analysis groups were comparable forall demographic and baseline characteristics variables.

Having demonstrated the clinical meaningfulness of consistently improvedwalking speeds during the double-blind as a criterion forresponsiveness, the question of the magnitude of benefit becomes ofinterest. The fampridine non-responders, although providing no relevantefficacy information, do provide safety information regarding thoseindividuals who are treated with fampridine but show no apparentclinical benefit. As such, responder analyses of these groups wereperformed.

With respect to magnitude of benefit, FIG. 10 and Table 12 belowsummarizes the percent changes in walking speed at each double-blindvisit by responder analysis grouping. The mean improvement for thefampridine responders during the double-blind across 14 weeks oftreatment ranged from 24.6% to 29.0% compared to 1.7% to 3.7% for theplacebo group; this was highly significant (p<0.001) at every visit.Although providing no relevant efficacy information, results for thefampridine non-responders are also illustrated and show that there was,and could be, some worsening in walking speeds after 12-weeks when anon-responder is treated with fampridine. The improvement was stable(±3%) across 14 weeks of treatment, and was associated with improvementin two global measures (Subject Global Impression and Multiple SclerosisWalking Scale-12). The four placebo responders showed a 19% improvementin walking speed but there were too few subjects in this group formeaningful statistical comparison. Response status was not significantlyrelated to baseline demographics, including type or severity of MS.Adverse events and safety measures were consistent with previousexperience for this drug.

TABLE 12 Summary of percent change in Walking Speed at each double-blindvisit by responder analysis grouping. Summary Statistics Over Time Studyday Treatment titration 1st stbl 2nd stbl 3rd stbl Placebo Mean 1.7 2.61.8 3.7 (SEM) (2.21) (3.23) (3.11) (3.38) N# 47 46 46 45 Fampridine Mean8.3 3.5 −0.2 −6.5 Non-responders (SEM) (2.05) (1.90) (1.76) (2.49) N 9794 93 89 Fampridine Mean 27.4 24.6 29.0 27.3 Responders (SEM) (2.43)(2.44) (4.31) (3.52) N 58 58 57 58 FR vs. Placebo p-value{circumflexover ( )} <0.001 <0.001 <0.001 <0.001 FR vs. FNR p-value{circumflex over( )} <0.001 <0.001 <0.001 <0.001 FNR vs. PBO p-value{circumflex over( )} 0.080 0.884 0.497 0.022 ABBREVIATIONS: FR = Fampridine Responders;FNR = Fampridine Non-responders. #The treatment sample sizes presentedat individual time points may be smaller than those in the ITTpopulation due to dropouts or missed assessments. #The treatment samplesizes presented in the figure legend represent the number of ITTsubjects. Sample sizes at individual time points may be smaller due todropouts or missed assessments. {circumflex over ( )}P-values fromt-tests of the least-squares means using the mean square error via anANOVA model with effects for responder analysis grouping and center.

FIG. 11 and Table 13 summarize the changes in LEMMT at each double-blindvisit by responder analysis grouping. The mean improvement for thefampridine responders during the double-blind ranged from 0.09 to 0.18units compared to −0.04 units at each visit for the placebo group; thiswas significant at every visit except the second stable dose visit(p=0.106). Although providing no relevant efficacy information, resultsfor the fampridine non-responders are also illustrated and show thatthere was, and could be, some significant improvement in leg strengthwhen non-responder is treated with fampridine. This suggests thatalthough a clinically meaningful response can be linked to about 37% ofsubjects treated with Fampridine-SR, additional subjects may havefunctional improvements on variables other than walking speed.

TABLE 13 Summary of percent change in LEMMT at each double-blind visitby responder analysis grouping. Summary Statistics Over Time Study dayTreatment titration 1st stbl 2nd stbl 3rd stbl Placebo Mean −0.04 −0.04−0.04 −0.04 (SEM) (0.035) (0.042) (0.039) (0.042) N# 46 46 46 45Fampridine Mean 0.12 0.10 0.09 0.10 Non-responders (SEM) (0.028) (0.033)(0.036) (0.038) N 95 94 94 89 Fampridine Mean 0.18 0.09 0.09 0.17Responders (SEM) (0.029) (0.032) (0.043) (0.045) N 58 58 58 58 FR vs.Placebo p-value{circumflex over ( )} <0.001 0.023 0.106 0.004 FR vs. FNRp-value{circumflex over ( )} 0.178 0.627 0.739 0.311 FNR vs. PBOp-value{circumflex over ( )} <0.001 0.003 0.038 0.032 ABBREVIATIONS: FR= Fampridine Responders; FNR = Fampridine Non-responders. #The treatmentsample sizes presented at individual time points may be smaller thanthose in the ITT population due to dropouts or missed assessments.Treatment sample sizes presented in the figure legend represent thenumber of ITT subjects. Sample sizes at individual time points may besmaller due to dropouts or missed assessments. {circumflex over( )}P-values from t-tests of the least-squares means using the meansquare error via an ANOVA model with effects for responder analysisgrouping and center.

FIG. 12 and Table 14, below, summarize the changes in Overall AshworthScore at each double-blind visit by responder analysis grouping. Themean reduction from baseline (indicative of improvement) for thefampridine responders during the double-blind ranged from −0.18 to −0.11units compared to −0.11 to −0.06 for the placebo group. The fampridineresponders were numerically superior to placebo but there wasinsufficient evidence to detect significant differences. Althoughappearing to provide little relevant efficacy information, results forthe fampridine non-responders are also illustrated.

TABLE 14 Summary of change in overall Ashworth score at eachdouble-blind visit by responder analysis grouping. Summary StatisticsOver Time Study day Treatment titration 1st stbl 2nd stbl 3rd stblPlacebo Mean −0.06 −0.11 −0.06 −0.13 (SEM) (0.069) (0.073) (0.070)(0.073) N# 46 46 46 45 Fampridine Mean −0.16 −0.08 −0.07 0.00Non-responders (SEM) (0.044) (0.053) (0.054) (0.056) N 95 94 94 89Fampridine Mean −0.14 −0.18 −0.11 −0.18 Responders (SEM) (0.058) (0.066)(0.060) (0.055) N 58 58 58 58 FR vs. Placebo p-value{circumflex over( )} 0.343 0.374 0.717 0.680 FR vs. FNR p-value{circumflex over ( )}0.675 0.210 0.911 0.064 FNR vs. PBO p-value{circumflex over ( )} 0.1510.823 0.772 0.189 ABBREVIATIONS: FR = Fampridine Responders; FNR =Fampridine Non-responders. #The treatment sample sizes presented atindividual time points may be smaller than those in the ITT populationdue to dropouts or missed assessments. {circumflex over ( )}P-valuesfrom t-tests of the least-squares means using the mean square error viaan ANOVA model with effects for responder analysis grouping and center.

Adverse events most commonly reported prior to treatment were accidentalinjury, reported by 12 (5.8%) subjects, nausea, reported by 9 (4.4%)subjects, and asthenia, diarrhea, and paresthesia, each reported by 8(3.9%) subjects. Six (2.9%) subjects also reported headache, anxiety,dizziness, diarrhea, and peripheral edema. These adverse events areindicative of the medical conditions affecting people with MS.

Conclusions. The data does not appear to support either a number ofanecdotal reports or expectations from preclinical pharmacology thatdoses higher than about 10 to 15 mg b.i.d., and even about 10 mg b.i.d.,should be associated with greater efficacy. The data presented below inTable 15 support this, based on the new responder analysis methodology.

TABLE 15 Comparison of 10 mg vs. 15 mg among Responders 10 mg 15 mg (N =51) (N = 50) Responders N (%) 18 (35.3) 18 (36.0) Average % CFB in WalkSpeed: 27.6% (18.39)  29.6% (22.43)  Mean (SD) % Change in Walk Speed byVisit: 26%-32% 27%-31% minimum-maximum Average SGI 4.8 (1.09)  4.7(1.09)  Average Change in MSWS-12* −11.1 (21.9)   −7.8 (19.6)   *For theaverage change in the MSWS-12, a negative score is indicative ofsubjective improvement.

A responder analysis based on consistency of improvement provides asensitive, meaningful approach to measuring effects on the timed 25 footwalk and may be used as a primary endpoint for future trials. This datasuggest that for responsive subjects (approximately 37%), treatment withfampridine at doses of 10-20 mg bid produces substantial and persistentimprovement in walking.

Efficacy.

There are no notable differences between 10 mg bid and 15 mg bid amongsubjects who respond to drug. In fact, the largest difference, favorsthe 10 mg bid group (see MSWS-12 result).

Safety.

With respect to safety, there are three considerations: There was anapparent decline below baseline walking speed at the last visit on drugin the fampridine non-responders in the 10 mg bid and 20 mg bid groups,but not the 15 mg bid group. This may or may not be significant, but isnot clearly dose related. There was an apparent rebound effect, withwalking speed dropping below baseline, among fampridine treated subjectsat the two week follow-up visit; this occurred in the 15 and 20 mg butnot the 10 mg bid group. Serious AE's were more frequent in the 15 mgand 20 mg bid groups 10% and 12% rates vs. 0% rate in 10 mg bid and 4%in placebo groups. This may or may not be significant, but the risk ofpotentially related SAEs, particularly seizures appears to bedose-related from all available data and based on mechanism of action.Based on this data, it would appear that a 10 mg bid dose is preferredbecause of its favorable risk to benefit ratio compared with the 15 and20 mg doses.

Although the present invention has been described in considerable detailwith reference to certain preferred embodiments thereof, other versionsare possible. Therefore the spirit and scope of the appended claimsshould not be limited to the description and the preferred versionscontain within this specification.

1. A method of increasing walking speed comprising administering to apatient with multiple sclerosis an effective amount of a sustainedrelease aminopyridine composition twice daily, wherein said effectiveamount is less than about 15 milligrams of aminopyridine.
 2. The methodof claim 1, wherein said effective amount is about 10 to about 15milligrams of aminopyridine.
 3. The method of claim 1, wherein saideffective amount is about 10 milligrams of aminopyridine.
 4. The methodof claim 1, wherein said aminopyridine is 4-aminopyridine.
 5. The methodof claim 4, wherein said sustained release 4-aminopyridine compositionexhibits a C_(max):C_(r) ratio from about 1.0 to 3.5.
 6. The method ofclaim 1, wherein said twice daily administration comprises. about every12 hours.
 7. A method of improving lower extremity muscle tonecomprising administering to a patient with multiple sclerosis aneffective amount of a sustained-release aminopyridine composition twicedaily, wherein said effective amount is less than about 15 milligrams ofaminopyridine.
 8. The method of claim 7, wherein said effective amountis about 10 to about 15 milligrams of aminopyridine.
 9. The method ofclaim 7, wherein said effective amount is about 10 milligrams ofaminopyridine.
 10. The method of claim 7, wherein said aminopyridine is4-aminopyridine.
 11. The method of claim 10, wherein said sustainedrelease 4-aminopyridine composition exhibits a C_(max):C_(r) ratio fromabout 1.0 to 3.5.
 12. A method of improving lower extremity musclestrength comprising administering to a patient with multiple sclerosisan effective amount of a sustained-release aminopyridine compositiontwice daily, wherein said effective amount is less than about 15milligrams of aminopyridine.
 13. The method of claim 12, wherein saideffective amount is about 10 to about 15 milligrams of aminopyridine.14. The method of claim 12, wherein said effective amount is about 10milligrams of aminopyridine.
 15. The method of claim 12, wherein saidaminopyridine is 4-aminopyridine.
 16. The method of claim 15, whereinsaid sustained release 4-aminopyridine composition exhibits aC_(max):C_(r) ratio from about 1.0 to 3.5. 17-20. (canceled)